Association of Polymorphisms in miR146a, an Inflammation-Associated MicroRNA, with the Risk of Idiopathic Recurrent Spontaneous Miscarriage: A Case-Control Study

It has been established that microRNAs (miRNAs) are involved in the regulation of immune responses and serve as biomarkers of inflammatory diseases as well as recurrent spontaneous miscarriage (RSM). Herein, we aimed to study the relationship between three functional miR146a gene polymorphisms with idiopathic RSM (IRSM) susceptibility. We recruited 161 patients with IRSM and 177 healthy women with at least one live birth and without a history of abortion. Genotyping was performed using RFLP-PCR and ARMS-PCR methods. We found that the rs6864584 T/C decreased the risk of IRSM under dominant TT+TC vs. CC (OR = 0.029) and allelic C vs. T (OR = 0.028) contrast models. Regarding rs2961920 A/C and rs57095329 A/G polymorphisms, the enhanced risk of IRSM was observed under different genetic contrasted models, including the codominant CC vs. AA (OR = 2.81 for rs2961920) and codominant GG vs. AA (OR = 2.36 for rs57095329). After applying a Bonferroni correction, haplotype analysis revealed a 51% decreased risk of IRSM regarding the ACA genotype combination. This is the first study reporting that miR146a rs57095329 A/G, rs2961920A/C, and rs6864584 T/C polymorphisms are associated with the risk of IRSM in a southern Iranian population. Performing replicated case-control studies on other ethnicities is warranted to outline the precise effects of the studied variants on the risk of gestational trophoblastic disorders.


Introduction
Recurrent spontaneous miscarriage (RSM) is defined as the occurrence of 3 consecutive losses < 20 weeks gestation [1]. This condition has a fascinating yet poorly understood etiology. It affects about 5% of couples; due to its complex and often undefined etiology, RSM has been a tremendous challenge in reproductive medicine [1,2]. Exposure to environmental factors, genetics and hormonal abnormalities, immune reactions, embryo-fetal infections, uterine pathologies, antiphospholipid syndrome, sperm oxidative stress, drug abuse, and psychological trauma have been implicated as etiologic factors of RSM [3][4][5]. However, 40-60% of the RSM cases have no specific pathology and thus are classified as unexplained or idiopathic RSM (IRSM) [6]. Lately, several single nucleotide polymorphisms (SNPs) have been associated with IRSM occurrence [7][8][9]. Of those, SNPs that resided in genes encoding microRNAs (miRNAs or miRs) have been brought to the attention [10]. Essentially, a significant proportion of IRSM might be due to SNPs in coding-or noncoding genome [11].
For all we know, no study has integrated these findings into the relationship between these SNPs and IRSM. Hence, in this preliminary study, we aimed to examine the relationship between three miR-146a SNPs (rs2961920A/C, rs57095329 A/G, and rs6864584 T/C) with IRSM incidence in a south-east Iranian population.

Subjects and Methods
2.1. Participants. The study group consisted of 161 women with idiopathic IRSM, who presented with a history of at least two consecutive pregnancy losses at less than 20 weeks of gestation and 177 healthy women as controls. The enrollment procedure has been described in our previous work [29]. The local Ethics Committee of Zahedan University of Medical Sciences approved the study (IR.ZAUMS.-REC.1399.093). Informed consent was taken from all participants.
2.2. DNA Extraction and Genotyping. The salting-out method was employed to extract DNA from the whole venous blood of participants. A NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) was used to verify the quantity and purity of the extracted DNAs. Electrophoresis on agarose gel was carried out to evaluate the quality of extracted DNA.
Genotyping of rs6864584 polymorphisms was performed via both polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The used primer sequences were already reported in Sargazi et al.'s study [30]. For detecting all three SNPs, PCR was performed in a total volume of 15 μL, containing 1 μL (100 ng/μL) genomic DNA, 1 μL of each primer (6pmol), 10μL Taq DNA Polymerase 2X Master Mix Red (Ampliqon, Denmark), and 2 μL of distilled water. The PCR conditions were as follows: denaturation at 95°C for 5 min, 35 cycles at 95°C for 35 s, annealing temperature (according to Table 1 for each SNP) for 30 s and 72°C for 45 s, and a final extension at 72°C for 5 min. PCR products were electrophoresed on 1.5% agarose gel and stained in 0.25 μg/mL ethidium bromide, and bands were visualized ( Figure 1). The C allele of rs6864584 was digested by the ApaI and produced a 152-and 148 bp band, and the undigested T allele has a 300 bp fragment. Regarding rs57095329 and rs2961920, we used allele-specific amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) [31] in two well-defined conditions. For this purpose, the forward primer for one allele and the common primer were placed in one well. At the same time, the other forward primer specific for the other allele and the common primer were added to another well, and PCR reaction was performed following the condition above. In the presence of either the homozygous genotypes, the amplified band will be visualized in separate wells. In contrast, the bands can be visualized in both wells in the presence of a heterozygous genotype. The amplicon sizes for genotyping rs57095329 and rs2961920 were 153 and 221 bp, for both A and G or A and C alleles, respectively ( Table 1). The bands were then displayed via agarose gel electrophoresis ( Figure 1). About 30% of the samples were randomly regenotyped for quality control, and the reproducibility of the results was 100%.
2.3. Statistical Analysis. The SPSS 23.0 software (IBM Corporation, Armonk, NY) was used for data analysis. Deviations from Hardy Weinberg equilibrium (HWE) were examined using a chi-square test. Fisher's exact test was used to compare differences between data sets. The association between the alleles and genotypes of miR146a polymorphisms and IRSM risk was examined by odds ratios (ORs) and 95% confidence intervals (CIs), using unconditional logistic regression analysis. The SHEsis software platform 2 Disease Markers  3 Disease Markers was used to determine the amount of linkage disequilibrium (LD) between the studied SNPs [32]. Statistical significance considered as a p value < 0.05.

Results
The subject's ages differed from 15 to 55 years, with the mean age of 31:6 ± 6:8 (in controls) and 32:5 ± 6:6 (in IRSM patients). The mean (SD) BMI of controls was 26:2 ± 4:4 versus 26:3 ± 4:7 in cases. We found no marked difference between cases and controls regarding age (p = 0:23) and body mass index (BMI, p = 0:77). For all the studied SNPs, we did not observe any departure from HWE in healthy women (p value for HWE > 0:05 as shown in Table 2), indicating the proper selection of the controls.
Genotypic and allelic distribution of miR146a SNPs is presented in Table 2. We found that the AC genotype of rs2961920, the TC genotype of rs6864584, and the AG genotype of rs57095329 were more frequent in controls and selected as the reference genotypes. Both rs57095329 A/G and rs2961920 A/C enhanced the risk of IRSM in our population, whereas alleles and genotypes of rs6864584 C/T showed a protective role against the disease. The C allele of  Table 3 shows the interaction analysis of miR146a polymorphisms on IRSM risk. None of the genotype combinations of rs2961920, rs6864584, and rs57095329 polymorphisms significantly affected IRSM risk, after applying a Bonferroni correction for multiple testing (p > 0:017). Table 4 demonstrates the haplotype analysis of miR146a SNPs in IRSM patients and healthy women. Our results indicated that the ACA haplotype significantly decreased the risk of IRSM compared with rs2961920A, rs6864584T, and rs57095329T haplotype (OR ð95%CIÞ = 0:49 ð0:28 -0:85Þ, p value = 0.01).
Linkage disequilibrium (LD) analysis indicated that the cohesion coefficient between rs2961920A/C and rs57095329 A/G was higher than the others. Low D' percentages were noticed between rs6864584 C/T and rs57095329 A/G and also between rs6864584 C/T and rs2961920 A/C polymorphisms (about 5%). Hence, our results suggest no strong linkage between the studied variants in our population ( Figure 2). We used the WebLogo server to determine the conservation of DNA sequences containing the studied polymorphism sites. As illustrated in Figure 3, miR146a rs57095329 A/G and rs2961920 A/C polymorphisms are not located in the conserved regions across multiple mammalian species.

Discussion
miR146a has a pivotal role in modulating inflammatory responses, as it negatively modulates the inflammatory cytokines, including interleukin 6 (IL-6) and interleukin 6 (IL-8) [33], T-helper 17 (Th17) [34], T-helper 1 (Th1) [35], and NF-κB pathway [36]. Genetic variations in the miR146a gene have been correlated with the risk of different diseases. We had already shown that different haplotypes and genotypes of miR146a polymorphisms could influence the odds of developing chronic kidney disease, another inflammatory condition [30]. However, no study has reported the link between these functional miR146a polymorphisms and IRSM in the Iranian population. The current study's findings demonstrated that alleles and genotypes of miR146a SNPs were associated with the risk of IRSM. Besides, we found that a haplotype of miR146a polymorphisms decreased the disease risk by 51%. Our results suggest that miR146a might be a biomarker for IRSM susceptibility.
As the first report on the Iranian population, Alipour and coworkers demonstrated an association between an SNP located in miR146a and IRSM risk in 2019. In this connection, they screened the rs2910164 C/G polymorphism on 120 Iranian women diagnosed with IRSM and 90 controls. They discovered significant associations between this SNP and RSM incidence in CC vs. GG (OR = 0:481, p = 0:015) and CG vs. GG (OR = 2:125, p = 0:014) genetic models. Besides, individuals who had either heterozygous or homozygous specified polymorphism (carriers of CG+GG genotype) had a 2.077-fold increased RSM risk [48]. The findings of this report have established the role of miR146a, as a circulating RNA, and SNPs located in miR146a in the pathophysiology of IRSM. Two years later, Babakhanzadeh et al. failed to report an association between CC vs. CG +GG (p = 0:854) and CC+CG vs. GG (p = 0:282) genotypes of miR146a rs2910164 polymorphism and risk of IRSM in women from Yazd Province, located in the center of Iran [49]. Other SNPs in the miR146a gene, rs6864584 T/C, rs2961920 A/C, and rs57095329 A/G, were less studied. Still, the findings of some studies support the contribution of these variants to susceptibility to different diseases. Zha et al. found a significant correlation between allele and genotypes of miR146a rs57095329 A/G polymorphism and the risk of developing coronary artery lesions in Kawasaki patients. Meanwhile, the miR146a rs6864584 T/C was not correlated with the risk of developing Kawasaki disease [50]. In another case-control study, Devanandan et al. proposed that the A rs57095329 G rs2910164 enhances the risk of developing acute lymphoblastic leukemia by fold, whereas the G rs57095329 G rs2910164 represented a protective haplotype for the disease [51].
To this date, no study has reported the possible correlation between these two miR146a SNPs with the risk of gestational trophoblastic diseases. There are very limited reports on the correlation between miR146a rs2961920 A/C and the risk of a multifactorial disease. In this context, Guo

Disease Markers
and associates genotyped 502 healthy controls and 529 patients diagnosed with glioma using the Agena MassARRAy Gold technique. Their findings revealed that rs2961920, as a promoteric variant for miR146a and an intronic variant for MIR3142HG, enhanced the glioma susceptibility in Chinese subjects (OR = 1:53, p = 0:019) [52]. In another study, Oetting et al. showed that the rs2961920, a proxy for rs2910164, can independently predict acute rejection in kidney transplant recipients [53]. In agreement with these reports, we found the enhanced risk of IRSM in Iranian subjects carrying the CC genotype of miR146a rs2961920 A/C under codominant CC vs. AA, recessive CC vs. AC+AA, and allelic C vs. A mode of inheritance.
As a circulating miRNA, miR146a controls megakaryocyte differentiation, adaptive immunity, and cancer [54]. On the other hand, completion of pregnancy highly depends on maternal immune tolerance to the fetal-placental unit, where imbalances between some signaling molecules (specifically Th1-and Th2-type cytokines) increase the incidence of miscarriage [55].
The decidual tissue contains different cytokineproducing cells [56]. Zhao et al. showed that miR146a-5p is downregulated in the deciduae of RSM patients compared with healthy controls [55]. Moreover, it has been established that miR146a improves the decidual cytokine microenvironment through modulating the Toll-like receptor (TLR) pathway and other possible mechanisms of RSM pathogenesis [57]. Furthermore, Chen et al. proposed that miR146a downregulates TLR4, tumor necrosis factor receptor-associated factor-6 (TRAF6), and IL-1 receptor-associated kinase 1 (IRAK1) and phosphorylates NF-κB. Consequently, suppressing TLR4/TRAF6/JNK and TLR4/NF-κB pathways inhibits proinflammatory cytokine production [58]. This is important since the upregulation of NF-κB and/or TLR4 at the protein or gene level is involved in RSM pathogenesis [59,60]. Gysler and colleagues reported that transfection of trophoblastic cells with miR146a-5p mimic caused increased IL8 secretion. However, in contrast, patients with adverse pregnancy outcomes express higher levels of miR146a-3p than healthy pregnant women experiencing no pregnancy complications [61]. Together, TLR4-and NF-κB-dependent miR-146a expression might contribute to the protective role of miR-146a polymorphisms against IRSM.
RSM is a very heterogeneous disease, and various etiologies are implicated in RSM, including factors known to be causative and those implicated as possible causative agents [i.e., genetic causes (aneuploidy, somatic mutations, and SNPs) and immunologic causes]. Autoimmune and alloimmune causes, anatomic causes, uterine Müllerian anomalies, infectious causes, environmental causes, smoking, endocrine factors, diabetes mellitus, and hematologic disorders are the common etiologic factors for recurrent abortion [62][63][64][65][66]. Dysregulation of many genes, including miR146a, is important in preserving pregnancy; therefore, SNPs within these genes might predict RSM susceptibility. Although the etiology of RSM is still unclear, discovering candidate SNPs can help understand the pathophysiology of this complex disease. Still, other mechanisms are involved in the pathogenesis of IRSM that remains to be fully elucidated. Such   Disease Markers DNA variations might serve as a valuable prognostic factor to predict the risk of developing such gestational diseases and can be exploited in "Personalized Medicine" applications [67,68]. Despite all the efforts made, there were some limitations to the current study. The main limitation was using a relatively small sample size. Although we could not perform sequencing to validate our results, random regenotyping of samples showed a very high genotyping accuracy. Moreover, an assessment of different hormones in the serum samples of IRSM patients and healthy controls is needed to correlate miR-146a with serum hormones and give a better idea of the clinical significance of this circulating miRNA. Finally, mRNA levels of miR146a were not evaluated to determine the link between genotypes of miR146a polymorphisms and miR146a expression, which requires further study.

Conclusion
This study first reported that miR146a rs57095329 A/G, rs2961920 A/C, and rs6864584 T/C polymorphisms are correlated with the risk of IRSM in a southern Iranian population. These results provide an opportunity to approach the problems of prognosis and diagnosis of RSM. Still, performing replicated case-control studies on other ethnicities is warranted to outline the precise effects of the studied variants on the risk of gestational disorders.

Data Availability
Derived data supporting the findings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
The local Ethics committee of Zahedan University of Medical Sciences approved the study (Ethical code: IR.ZAUMS.-REC.1399.093) (the webpage of ethical approval code is available at https://ethics.research.ac.ir/EthicsProposalViewEn .php?id=138328).

Consent
Informed consent was taken from all participants.

Conflicts of Interest
The authors declare that they have no conflicts of interest.

Authors' Contributions
SS and SS contributed to methodology and writing the draft, BM, MM, SS, and SM contributed to genotyping, MHN carried out data analysis, MG contributed to clinical patient assessment, and SS contributed to supervision and editing.